Rafael Salto

Real-time phosphate sensing in living cells using fluorescence lifetime imaging microscopy (FLIM).

Phosphate ions play important roles in signal transduction and energy storage in biological systems. However, robust chemical sensors capable of real-time quantification of phosphate anions in live cells have not been developed. The fluorescein derivative dye 9-[1-(2-methyl-4-methoxyphenyl)]-6-hydroxy-3H-xanthen-3-one (2-Me-4-OMe TG) exhibits the characteristic excited-state proton-transfer (ESPT) reaction of xanthenic derivatives at approximately physiological pH resulting in the dependence of the dyes nanosecond fluorescence decay time on the phosphate buffer concentration. This allows the 2-Me-4-OMe TG dye to be used with fluorescence lifetime imaging microscopy (FLIM) as a real-time phosphate intracellular sensor in cultured cells. This methodology has allowed the time course of cellular differentiation of MC3T3-E1 murine preosteoblast cells to be measured on the basis of the decrease in the decay time of 2-Me-4-OMe TG. These changes were consistent with increased alkaline phosphatase activity in the extracellular medium as a marker of the differentiation process.

β-Hydroxy-β-Methylbutyrate (HMB) Normalizes Dexamethasone-Induced Autophagy-Lysosomal Pathway in Skeletal Muscle

Dexamethasone-induced muscle atrophy is due to an increase in protein breakdown and a decrease in protein synthesis, associated with an over-stimulation of the autophagy-lysosomal pathway. These effects are mediated by alterations in IGF-1 and PI3K/Akt signaling. In this study, we have investigated the effects of β-Hydroxy-β-methylbutyrate (HMB) on the regulation of autophagy and proteosomal systems. Rats were treated during 21 days with dexamethasone as a model of muscle atrophy. Co-administration of HMB attenuated the effects promoted by dexamethasone. HMB ameliorated the loss in body weight, lean mass and the reduction of the muscle fiber cross-sectional area (shrinkage) in gastrocnemius muscle. Consequently, HMB produced an improvement in muscle strength in the dexamethasone-treated rats. To elucidate the molecular mechanisms responsible for these effects, rat L6 myotubes were used. In these cells, HMB significantly attenuated lysosomal proteolysis induced by dexamethasone by normalizing the changes observed in autophagosome formation, LC3 II, p62 and Bnip3 expression after dexamethasone treatment. HMB effects were mediated by an increase in FoxO3a phosphorylation and concomitant decrease in FoxO transcriptional activity. The HMB effect was due to the restoration of Akt signaling diminished by dexamethasone treatment. Moreover, HMB was also involved in the regulation of the activity of ubiquitin and expression of MurF1 and Atrogin-1, components of the proteasome system that are activated or up-regulated by dexamethasone. In conclusion, in vivo and in vitro studies suggest that HMB exerts protective effects against dexamethasone-induced muscle atrophy by normalizing the Akt/FoxO axis that controls autophagy and ubiquitin proteolysis.

Internalization of the Receptor for Advanced Glycation End Products (RAGE) is Required to Mediate Intracellular Responses

To dissect the rat receptor for advanced glycation end products (RAGE) subcellular distribution and trafficking in eukaryotic cells, an expression system coding for a fusion protein between the RAGE and an enhanced green fluorescent protein (EGFP) has been used. The RAGE-EGFP protein is expressed at the plasma membrane of CHO-k1 and Neuro-2a (N2a) cells and retains the capacity to bind Texas Red-labelled advanced glycation end products (AGEs). AGEs addition to the cell cultures induced a change in the subcellular distribution of the fluorescent RAGE-EGFP protein compatible with an internalization of the AGEs-RAGE complex. Furthermore, while N2a cells expressing the RAGE-EGFP showed an increase in ERK1/2 phosphorylation and NF-kappaB DNA binding in response to AGEs, pre-incubation with dansyl-cadaverine or phenylarsine oxide, inhibitors of receptors internalization, blocked the activation of ERKs and other intracellular responses mediated by AGEs. These results suggest that internalization plays a key role in the signal transduction mediated by RAGE.

Modulation of glucose transporters in rat diaphragm by sodium tungstate

Oral administration of sodium tungstate is an effective treatment for diabetes in animal models. We examined the effects of 6 weeks of oral administration of tungstate on glucose transporters (GLUT) in streptozotocin‐induced diabetic rat diaphragm. Diabetes decreased GLUT4 expression while tungstate treatment normalized not only GLUT4 protein but also GLUT4 mRNA in the diabetic rats. Furthermore, treatment increased GLUT4 protein in plasma and internal membranes, suggesting a stimulation of its translocation to the plasma membrane. Tungstate had no effect on healthy animals. There were no differences in the total amount of GLUT1 transporter in any group. We conclude that the normoglycemic effect of tungstate may be partly due to a normalization of the levels and subcellular localization of GLUT4, which should result in an increase in muscle glucose uptake.

Conversion of leucine to β-hydroxy-β-methylbutyrate by α-keto isocaproate dioxygenase is required for a potent stimulation of protein synthesis in L6 rat myotubes

L‐Leu and its metabolite β‐hydroxy‐β‐methylbutyrate (HMB) stimulate muscle protein synthesis enhancing the phosphorylation of proteins that regulate anabolic signalling pathways. Alterations in these pathways are observed in many catabolic diseases, and HMB and L‐Leu have proven their anabolic effects in in vivo and in vitro models. The aim of this study was to compare the anabolic effects of L‐Leu and HMB in myotubes grown in the absence of any catabolic stimuli.

The glucose-lowering agent sodium tungstate increases the levels and translocation of GLUT4 in L6 myotubes through a mechanism associated with ERK1/2 and MEF2D.

Aims/hypothesisThe aim of this study was to investigate the action of the glucose-lowering compound sodium tungstate on glucose transport in muscle myotubes and to unravel the molecular events underlying the effects observed.MethodsWe studied the effects of tungstate on 2-deoxy-d-glucose uptake, levels and translocation of the glucose transporters GLUT4 and GLUT1, and Glut4 (also known as Slc2a4) promoter activity. We also measured the modifications of individual components of the signalling pathways involved in the effects observed.ResultsTungstate increased 2-deoxy-d-glucose uptake in differentiated L6 myotubes through an increase in the total amount and translocation of GLUT4 transporter. The effects on glucose uptake were additive to those of insulin. Tungstate activated transcription of the Glut4 promoter, as shown by an increase in Glut4 mRNA, and by a promoter reporter assay. The assay of deletions of the Glut4 promoter indicated that the effect of tungstate is mediated by the myocyte enhancer factor 2 (MEF2)-binding domain. Accordingly, MEF2 levels and DNA binding activities were increased in response to the treatment. Tungstate-induced glucose uptake and GLUT4 transcriptional activation were dependent on the activation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), while no changes were observed in the phosphorylation state of the β subunit of the insulin receptor, in the phosphatidylinositol 3-kinase pathway or in the activation of 5′AMP-activated protein kinase.Conclusions/interpretationTungstate activates glucose uptake in myotubes through a novel ERK1/2-dependent mechanism. This effect is exerted by an increase in the content and translocation of the GLUT4 transporter. This is the first report of a glucose-lowering compound activating Glut4 transcription through an ERK1/2-dependent increase in MEF2 levels.

Modulation of hepatic and intestinal glutathione S-transferases and other antioxidant enzymes by dietary lipids in streptozotocin diabetic rats.

Antioxidant enzymes in liver and small intestine were investigated using control and streptozotocin diabetic rats fed diets with 5% olive, sunflower or fish oil for five weeks. In liver, Glutathione Peroxidase and Superoxide Dismutase decreased and in intestine Glutathione-S-transferase (GST) increased by diabetes. In isolated jejunum and ileum, this increase in GST activity was due to an increase in GST-alpha and -mu isoenzymes in jejunum and GST-alpha, mu and -pi in ileum. Since GST plays an important role in protecting tissues from oxidative damage, our results highlight the role of the intestine against free radicals in physiological or pathological situations.

Increased Diaphragm Expression of GLUT4 in Control and Streptozotocin-Diabetic Rats by Fish Oil-Supplemented Diets

Dietary fat intake influences plasma glucose concentration through modifying glucose uptake and utilization by adipose and skeletal muscle tissues. In this paper, we studied the effects of a low-fat diet on diaphragm GLUT4 expression and fatty acid composition in control and streptozotocin-induced diabetic rats. Control as well as diabetic rats were divided into three different dietary groups each. Either 5% olive oil, 5% sunflower oil, or 5% fish oil was the only fat supplied by the diet. Feeding these low-fat diets for 5 wk induced major changes in fatty acid composition, both in control and in diabetic rats. Arachidonic acid was higher in diabetic olive and sunflower oil-fed rats with respect to fish oil-fed, opposite to docosahexaenoic acid which was higher in diabetic fish oil-fed rats with respect to the other two groups. Animals receiving a fish oil diet had the lowest plasma glucose concentration. GLUT4 expression in diaphragm, as indicated by GLUT4 protein and mRNA, is modulated both by diabetes and by diet fatty acid composition. Diabetes induced a decrease in expression in all dietary groups. Plasma glucose levels correlated well with the increased amount of GLUT4 protein and mRNA found in fish oil-fed groups. Results are discussed in terms of the influence that arachidonic and n−3 polyunsaturated fatty acids may exert on the transcriptional and translational control of the GLUT4 gene.

Evolution of pyruvate carboxylase and other biotin containing enzymes in developing rat liver and kidney

The evolution of pyruvate carboxylase has been studied in rat liver and kidney during perinatal development. The pyruvate carboxylase activity, amount of enzyme and mRNA levels have been assayed from 2 days before delivery to weaning. In liver, there is a peak of activity and amount of enzyme 24 h before delivery and 2 peaks, at 12 h and 6 days, after parturition. The transcription of the enzyme gene followed a similar pattern, with mRNA peaks preceding those of activity and amount of enzyme. However, in kidney, pyruvate carboxylase activity, amount and mRNA remain low until weaning. These results confirm the limited role of renal gluconeogenesis during the perinatal development. Since all carboxylases contain biotin as prosthetic group, the biotinylation of pyruvate carboxylase during the perinatal period was investigated by western-blot using streptavidin-biotin peroxidase. In the mitochondrial samples from liver and kidney, all the pyruvate carboxylase detected was fully biotinylated, indicating an early development of the holocarboxylase synthetase activity in the perinatal period. This Western-blot technique also allowed us the detection of other biotin-enzymes based on their molecular weight. In liver, during the perinatal development propionyl-coA and 3-methyl-crotonyl-coA carboxylases followed a pattern of induction similar to pyruvate carboxylase. In kidney, the expression of mitochondrial carboxylases was lower compared to liver and propionyl-coA carboxylase was not detected during the studied period

β-Hydroxy-β-Methylbutyrate (HMB) Promotes Neurite Outgrowth in Neuro2a Cells.

β-Hydroxy-β-methylbutyrate (HMB) has been shown to enhance cell survival, differentiation and protein turnover in muscle, mainly activating phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) and mitogen-activated protein kinases/ extracellular-signal-regulated kinases (MAPK/ERK) signaling pathways. Since these two pathways are related to neuronal survival and differentiation, in this study, we have investigated the neurotrophic effects of HMB in mouse neuroblastoma Neuro2a cells. In Neuro2a cells, HMB promotes differentiation to neurites independent from any effects on proliferation. These effects are mediated by activation of both the PI3K/Akt and the extracellular-signal-regulated kinases (ERK1/2) signaling as demonstrated by the use of specific inhibitors of these two pathways. As myocyte-enhancer factor 2 (MEF2) family of transcription factors are involved in neuronal survival and plasticity, the transcriptional activity and protein levels of MEF2 were also evaluated. HMB promoted MEF2-dependent transcriptional activity mediated by the activation of Akt and ERK1/2 pathways. Furthermore, HMB increases the expression of brain glucose transporters 1 (GLUT1) and 3 (GLUT3), and mTOR phosphorylation, which translates in a higher protein synthesis in Neuro2a cells. Furthermore, Torin1 and rapamycin effects on MEF2 transcriptional activity and HMB-dependent neurite outgrowth support that HMB acts through mTORC2. Together, these findings provide clear evidence to support an important role of HMB in neurite outgrowth.